Interest in tumor vaccines is increasing with recent announcements of promising clinical trials. Vaccine strategies partially rely on cytolytic T lymphocytes (CTLs) and natural killer (NK) cells to eliminate malignant cells by inducing rapid apoptosis. In part, these immune cells use death receptor ligands such as tumor necrosis factor-α (TNFα), FAS-L, and TRAIL to stimulate certain TNF-family death receptors on tumor target cells, resulting in activation of caspase-family proteases and triggering of apoptosis (Takeda et al., 2001; Montel et al., 1995a; Montel et al., 1995b; Sayers et al., 1998). Attempts to exploit these immune effector molecules as anticancer agents have resulted in early stage clinical trials that employ a recombinant soluble fragment of TRAIL and agonistic monoclonal antibodies targeting TRAIL receptors (Nagane et al., 2001). A limitation of such therapies, however, is acquired or intrinsic resistance to TNF-family death ligands and death receptors, which commonly occurs in advanced malignancies (Wuchter et al., 2001).
Metastasis is the primary cause of cancer-related mortality in patients with most forms of solid tumors (Jemal et al., 2006). Early-stage tumors remain relatively localized, due in part to dependence on cell anchorage to the extra-cellular matrix (ECM) (Kimura et al., 2001; Zhou et al., 2001; Rennebeck et al., 2005). Continued tumor progression gives rise to cells that can grow independent of anchorage to the ECM and may eventually metastasize to other tissues (Zhou et al., 2001; DeMarzo et al., 2003).
Metastasis is a multi-step process. First, metastatic cells must detach from their primary tumor and survive in an anchorage-independent manner. After detachment, cells must migrate to the lymphatic and circulatory systems while evading immune surveillance. Once in the circulation, cells must invade distal organs, implant within local tissues, and initiate de novo tumor growth. Although all of these steps are required for metastasis, anchorage-independent survival represents a critical stage in the development of metastatic disease (Mehlen et al., 2006; Glinsky et al., 1997).
Normally, cells undergo apoptosis upon detachment from their ECM, a self-initiated process termed “anoikis” (Frisch et al., 1994; Frisch et al., 2001). Recent studies in non-malignant epithelial and endothelial cells suggest that anoikis is mediated, in part, by activation of the death receptor pathway of caspase activation (Frisch et al., 2001). In this pathway, ligands bind to the extra-cellular domains of the TNF family of death receptors. Ligand binding results in the recruitment of the intra-cellular protein FADD to the receptor's cytoplasmic domain (Jin et al., 2005). Receptor-bound FADD then recruits procaspase 8 to form a death-inducing signalling complex (DISC) (Muzio et al., 1996; Chinnaiyan et al., 1995; Kischkel et al., 1995). Dimerization and self-cleavage of procaspase 8 within the DISC generates active caspase 8, which returns to the cytosol to activate effector caspases (Medema et al., 1997; Boatright et al., 2003). In non-malignant cells, detachment from the ECM induces the expression of the death receptor protein Fas and its ligand FasL, with resultant activation of caspase 8 in a FADD-dependent manner (Aoudjit et al., 2001; Rosen et al., 2002; Rytomaa et al., 2999; Frisch, 1999; Bachelder et al., 2001).
This mechanism for achieving caspase activation is referred to as the “extrinsic” pathway, standing in contrast to another apoptosis pathway that involves mitochondria, and which has been termed the “intrinsic” pathway (Schimmer et al., 2001). Stimuli that activate the intrinsic pathway include DNA damaging anticancer drugs, γ-irradiation, hypoxia, and growth factor deprivation, causing mitochondria to release cytochrome c and other apoptogenic proteins into the cytosol, resulting in caspase activation (Hajra and Liu, 2004).
Diverse mechanisms can create roadblocks to apoptosis within the extrinsic or intrinsic pathways, occurring commonly in many cancers during tumor progression and thus creating impediments to successful treatment. Documented resistance mechanisms relevant to the extrinsic pathway include reduced expression of TNF-family death receptors, shedding of soluble death receptors and expression of ligand-binding decoy receptors, reduced expression of caspases-8 and -10, and overexpression of intracellular caspase inhibitors (Wand and El-Deiry, 2003). Among the endogenous caspase inhibitors affecting the extrinsic pathway is c-FLIP, a protein resembling caspases-8 and -10, which can bind and prevent their activation at the DISC (Irmler et al., 1997; Scaffidi et al., 1999).
Thus, there is a need for molecules that restore sensitivity of tumor cells to TNF-family death receptors and so are useful therapeutic adjuncts to agents such as recombinant TRAIL and tumor vaccines.